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translation strategies Flashcards

1
Q

compare eukaryotic mRNAs to prokaryotic mRNAs

A

eukaryotic:
cap-5’UTR-AUG-ORF-Stop-3’UTR-PolyA

5’cap: binding of initiation factors

prokaryotic mRNA: polycistronic composition with shine dalgarno sequence and no cap

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2
Q

what is the function of the 5’ cap structure?

A

marks the 5’end of the first exon and aids in the splicing process:
-nucleo-cytoplasmic transport of mRNA via interaction with nuclear cap binding protein
-increases efficiency of translation by targeting formation of the preinitiation complex
-protects the transcript from 5’-3’ exoribonuclease activities
-blocks recognition of viral RNAs by an antiviral defense mechanism

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3
Q

name 3 standard ways cap structures of viral mRNAs are made

A

1.de novo synthesis by cellular enzymes: Adenoviridae, Retroviridae
2.synthesis by viral enzymes: Poxviridae
3.acquisition of preformed 5’ cap structures from cellular pre-mRNAs or mRNAs: Bunyviridae, Orthomoxyviridae

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4
Q

name unconventioanl viral capping pathways and which viruses employ these mechanisms

A
  1. rhabdoviridae-like: covalent enzyme-pNp-RNA intermediate with the nacent viral RNA –> transfer of GDP to RNA 5’ end –> 2’O methylation –> guanine N7 methylation
    2.Alphaviridae like: covalent enzyme-m7G intermediate: transfer of m7G to the RNA 5’ end
    3.Orthomyxoviridae-liek: EndoNs cap binding domain binds to cellular mRNA cap, viral RdRp continues nad produces viral RNA
    4.SARS CoV-2: RNAylation of nsp9 with viral RNA by NiRAN domain of nsp12 (RdRp) –> nsp9 covalently bound to viral RNA is replaced by GDP
    5.hepatitis C virus RNA is 5’capped with FAD
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5
Q

what are the functions of 3’processing of mRNA in eukaryotes?

A

stabilizes the mRNA
increases the efficiency of translation

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6
Q

name viral and cellular examples of mRNA without Poly A

A

viral: flaviviral, pestiviral, reoviral

cellular: histones

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7
Q

what is host cell shut off?

A

virus infection often results in modification of the host’s translational machinery to translate selectively the viral mRNAs

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8
Q

what are the special features of viral translation and why are they used?

A

why: maximize the coding capacity of their genomes of limited sizes

features:
1.functional polycistronic mRNA
2.readthrough of stop codons
3.polyproteins

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9
Q

how can multiple translation products be made from one primary transcript?

A

1.polyprotein processing
2.initiation at different start codons (reinitiation, leaky scanning, internal ribosomal entry, ribosomal shunting)
3.changing the translational reading frame (splicing, ribosomal frameshifting, RNA editing)
4.translational readthrough of stop codons

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10
Q

What is the ratelimiting step of translation?

A

initiation

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11
Q

what is required for translation and what is the result?

A

required: hydrolysis of ATP and GTP

results: formation of a complex containing the mRNA, the ribosome and the initiator Met-tRNA

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12
Q

Name and explain different ways for translation initiation

A

1.5’ end (cap) dependent initiation: EIF4F recognizes 5’ end, initiation complex binds to 5’cap and EIF4A scans in a 5’ to 3’ direction until initiating AUG is encountered, 40S subunit binds to eIF4G via eIF3

2.internal ribosome entry: initiation complex binds upstream of initiation codon

3.methionine-dependent initiation

4.methionine independent inititaion: RNA mimics tRNAi, can assemble 90S ribosomes without any eIFs or Met-tRNAi

5.IRES directed translation initiation: does not require the cap binding protein

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13
Q

how can translation be inhibited or modulated?

A

-missing 5’ cap structure
-strong RNA secondary structure in 5’ NTR
-long 5’NTR
-upstream AUG and open reading frames
-poor sequence context of the AUG codon (Kozak rule)

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14
Q

At which steps can viral infection interfere with eukaryotic mRNA translation at multiple steps?

A

-cleavage of eIF4G
-dephosphorylation of eIF4E
-modulation of 4E-BP
-IRES
-disruption of eIF2alpha phosphorylation
-stop codon recording
-reinitiation
-frameshifting
-elongation factor modification
-ribosome shunt
-leaky scanning

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15
Q

Explain translation of poliovirus and why 5’ end dependent initiation unlikely is

A

efficient translation of PV RNA despite the inhibition of host cell translation by the virus via eIF4G cleavage –> 5’end dependent translation unlikely
–>
direct ribosome binding to an internal RNA structure: IRES mediated, use of the c-terminal cleavage product after cleaving eIF4G

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16
Q

how can IRES activity be detected in the lab?

A

use of bicistronic plasmid with luciferase –> protein translation of circular RNA only possible with functional IRES element upstream of the ORF

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17
Q

how can the 40S ribosome be recruited to viral mRNA? what are structural features and initiation factor targets?

A

-5’cap
-cap stealing
-cap substitute (vpg)
-Type I - IV IRES
-3’CITE

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18
Q

for what and how can bicistronic expression vectors be used in biomedical research?

A

quantitive analysis of viral RNA replication by:
coupling expression of two genes driven by one promotor:
-gene+resistence gene
-gene with marker
–> selection of second-site mutations to study protein-protein interaction during virus replication

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19
Q

what is a bicsitronic expression vector?

A

coupled expression of two genes driven by one promotor

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20
Q

how can IRES insertions be used in virus research?

A

determination of precursor protein function in viral life cycle

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21
Q

what is polyprotein processing?

A

-strategy for producing multiple proteins from a single mRNA
-resulting polyprotein is proteolytically processed

22
Q

how is polyprotein processing achieved in picornavirus?

A

entire + sense RNA genome is translated into a single large protein –> processing by virus encoded proteases 2Apro and 3Cpro

23
Q

what is the advantage of polyprotein processing?

A

-gene expression can be controlled by the rate and extent of polyprotein processing
-alternative utilization of cleavage sites can produce proteins with different activity

24
Q

how is polyprotein processing achieved in Flavivirus?

A

RNA genome is translated into a polyprotein precursor processed by viral proteases and by host signal peptidases

25
Q

what are the hallmarks of pestiviral genome organization? Which Protein is essential?

A

-degree of NS2-3 cleavage is strain specific
-NS3 is essential for replication
-uncleaved NS2-3 essential for virion assembly
-NS2 autoprotease is activated by cellular chaperone DNAJC14/Jiv

26
Q

describe HCV Polyprotein processing

A

done by cellular and viral proteases:
-cleavage between NS2 and NS3 is catalyzed by an autoprotease in NS2
-uncleaved NS2-3 cannot be detected in cell culture and is not essential for virion morphogenesis
-the remainder of the cleavage sites in the HCV NS region are processed by a serine protease in NS3 and its cofactor NS4A

–>the NS2-3 cleavage is a prerequisite for functional viral replicase assembly by free NS3 (assembly of the viral replication complex on surface of NS3)

27
Q

how does polyprotein processing in FMDV work? Name 3 possible explanations

A

co-translation cleavage of short sequence of protease 2A:
-2A functions as a substrate for cellular proteinase (closely coupled to translation)
-2A sequence disrupts the normal peptide bond formation during translation
-2A sequence possesses an entirely novel type of proteolytic activity

28
Q

how does polyproteinprocessing work in alpha virus?

A

-proteolytic cleavage via nsP2
-switch from - strand synthesis to + strand synthesis

–>the fully cleaved viral replicase complex strongly favours production of subgenomic RNA

29
Q

What is the aim of reinitiation of translation?

A

producing two proteins from a single mRNA

30
Q

what does reinitiation of translation depend on?

A

-sequence context of the uORF AUG
-RNA secondary structure between uORF and downstream ORF
-distance between the two ORFs
-viral factors

31
Q

how can initiation of translation at different start codons be achieved?

A

-reinitiation
-leaky scanning
-internal ribosomal entry
-ribosomal shunting

32
Q

how does leaky scanning work in translation?

A

start of translation at the 2. AUG due to poor initiation context (Kozak rule) which leads to the ignoring of the first AUG

33
Q

how does ribosome shunting work?

A

start of translation at the 2. UAG due to the transport of the initiation complexes from the cap to AUG not linear along the mRNA –> non linear movement of the ribosome

34
Q

how can the reading frame of translation be changed?

A

-splicing
-ribosomal frameshifting
-RNA editing

35
Q

how does ribosomal frameshifting work?

A

ribosome can move to a different reading frame: RNA signals (slippery sequence followed by a pseudeknot or a stem loop

36
Q

how does ribosomal framehsifting work in retroviruses?

A

-ribosome pausing because of downstream pseudoknot/stem-loop structure
-slippage of 2 tRNAS into -1frame before/after petidyl transfer
-each tRNA pairs with the mRNA in the first 2 nucleotides of each codon
-3’ base of the 3’codon is available for binding of the next incoming tRNA
-free codon in mRNA resulting in -1 reading frame of the mRNA

37
Q

what is RNA editin?

A

mRNA sequence does not match the coding template in the viral genome

38
Q

how can mRNA be altered (RNA editing)?

A

alteration of the mRNA sequence by:
-insertion of additional nucleotides not specified by the template during synthesis
-posttrancriptional alteration of single bases in situ

39
Q

what is the potential of RNA editing?

A

-shift in the reading frame
-c-terminal extension (stop codon is changed)
-change in the amino acid sequence

40
Q

Explain posttrancriptional RNA editing of Hepatitis delta virus

A

adenosine desamination of A leads to I –> editing the stop codon –> +19 AS (longer antige)

41
Q

How does suppression of termination work in moloney murine leukemia vrius? what is the aim?

A

Amber stop codon (AUG –> CAG)
–>regulation of the relative ratio of Gag (structrural protein) and pol

42
Q

How does suppresion of termination work in Alphavirus?

A

Opal Stop codon (UGA) where the nucleotide context of UGAC (dwonstream C) is important for readthrough and suppresion of translation terminsation

43
Q

How can translation be regulatated by changing the host cell?

A

-host cell shut off
-interferon-induced cellular defense strategies
-viral defense mechanism

44
Q

How is the eIF4F complex inactivated by viral factors? Explain for different viruses

A

-Proteolysis: Poliovirus, Foot and mouth disease
-dephosphorylation of eIf4E: adenovirius, Influenza virus
-dephosphorylation of 4E-bp1: Poliovirus, Encephalomyocarditis virus

45
Q

What is the function of eIF4F complex?

A

-cap regonition
-binding to 40S subunit
-fusing of RNA secondary structures

46
Q

What are the components of the eIF4F complex?

A

eIF4E, eIF4G, eIF4A

47
Q

what is the effect of the proteolytic cleavage of eIF4G? What cleaves?

A

cleaving by viral proteases –> no eIF4E binding (no cap dependent initiation of translation) but IRES dependent translation

48
Q

how can viral and cellular RNA be circularized?

A

early infection: competition for translation factors: ccircularization via interaction of mRNA with eIF4G and PABP –> stimulates translation

late infection: release of translation factors –> no circularization

49
Q

how can host protein synthesis be inhibited by rota virus infection?

A

by jamming eIF4G-PABP interaction with rotavirus protein nsP3 –> cellular mRNA is released from eIF4G complex released –> viral mRNA can bind to complex via specifing binding to nsp3

50
Q

what is the interferon effect? where does it play a role?

A

regulation of translation by changing the host cell:
-virus cells release IFN alpha and beta
-IFN generate antiviral state in non-infected cells
-INF induces synthesis of protein kinase R
-viral dsRNA activates PKR –> blocks tranlsation initiation

Vilo (11 decks)

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